U.S. patent number 5,133,940 [Application Number 07/285,804] was granted by the patent office on 1992-07-28 for apparatus, for amplification of nucleic acids.
This patent grant is currently assigned to Orion Corporation Ltd.. Invention is credited to Nisse E. J. Kalkkinen, Hans E. Soderlund.
United States Patent |
5,133,940 |
Kalkkinen , et al. |
July 28, 1992 |
**Please see images for:
( Certificate of Correction ) ** |
Apparatus, for amplification of nucleic acids
Abstract
Amplification of nucleic acids is performed by incubating in a
polymerization vessel a reaction mixture which contains in a
suitable buffer solution, one or several single-stranded target
nucleic acids, suitable primers, deoxyribouncleoside triphosphates
and a polymerase. After a sufficient time for polymerication to
occur, the reaction mixture is transferred into another vessel for
the denaturation of the nucleic acids into single stranded nucleic
acids. After denaturation, the reaction mixture is transferred back
into the original vessel. The amplification process is regulated to
maintain a temperature advantageous for the action of the
polymerization enzyme in the polymerization vessel and a
temperature advantageous for denaaturation in the denaturation
vessel. Furthermore, the apparatus includes a liquid transfer
system for transferring the reaction liquid from one vessel to
another, which comprises at least one liquid transfer tube per
vessel pair which extends from within the polymerization vessel to
within the denaturation vessel.
Inventors: |
Kalkkinen; Nisse E. J. (Espoo,
FI), Soderlund; Hans E. (Espoo, FI) |
Assignee: |
Orion Corporation Ltd.
(FI)
|
Family
ID: |
8525619 |
Appl.
No.: |
07/285,804 |
Filed: |
December 16, 1988 |
Foreign Application Priority Data
Current U.S.
Class: |
422/138; 422/417;
422/203; 435/287.2; 422/208 |
Current CPC
Class: |
B01J
19/0046 (20130101); C12Q 1/686 (20130101); B01L
7/52 (20130101); C12Q 1/686 (20130101); C12Q
2527/149 (20130101); C12Q 2527/101 (20130101); B01J
2219/00722 (20130101); C40B 40/06 (20130101) |
Current International
Class: |
B01J
19/00 (20060101); B01L 7/00 (20060101); C08F
003/00 (); B10J 008/00 (); C12M 001/38 () |
Field of
Search: |
;435/290,316,513,819
;935/85-88 ;422/138,189,186.22,186.25,203,208,235,146,156
;137/571 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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|
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|
|
0200362 |
|
Mar 1986 |
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EP |
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0229701 |
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Jan 1987 |
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EP |
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0236069 |
|
Feb 1987 |
|
EP |
|
0237362 |
|
Mar 1987 |
|
EP |
|
Other References
Nucleic Acids Research vol. 16, No. 7, 1988 A Simple and Low Cost
DNA Amplifier by Rolio et al., pp. 3105 and 3106. .
DNA vol. 7, No. 6, 1988, pp. 441-447 Laboratory Methods-A
Programmable System to Perform the Polymerase Chain Reaction by
Heinz Ulrich and Joe W. Gray. .
Polymerase chain reaction automated at low cost by N. S. Foulkes et
al., pp. 5687-5688, Submitted May 17, 1988..
|
Primary Examiner: Housel; James C.
Assistant Examiner: Chan; William K. Y.
Attorney, Agent or Firm: Brumbaugh, Graves, Donohue &
Raymond
Claims
We claim:
1. An apparatus for use in amplification of nucleic acids
comprising at least one pair of reaction vessels, each pair of
vessels including a polymerization vessel and a denaturation
vessel; first thermoregulation means connected to the denaturation
vessel such that the denaturation vessel can be maintained at an
appropriate temperature for denaturation of double stranded nucleic
acids; second thermoregulation means connected to the
polymerization vessel such that the polymerization vessel can be
maintained at an appropriate temperature for polymerization of
nucleic acids; and a liquid transfer system which interconnects the
vessels by means of at least one liquid transfer tube extended from
within the denaturation vessel to within the polymerization vessel
and which is effective to transfer liquid form the denaturation
vessel to the polymerization vessel and from the polymerization
vessel to the denaturation vessel, said liquid transfer system
further comprising a pump with a reversible flow direction which in
combination with the liquid transfer tubes is effective to transfer
liquid between the vessels.
2. The apparatus according to claim 1, wherein the pump is
microprocessor controlled.
3. The apparatus according to claim 1, wherein the liquid transfer
system further comprises means for creating a pressure differential
between the polymerization vessel and the denaturation vessel such
that liquid is transferred between the vessels.
4. The apparatus according to claim 3, wherein the liquid transfer
system is connectable to a gas source for selectively introducing a
flow of a gas into one of the pair of reaction vessels by means of
at least one gas supply tube.
5. The apparatus according to claim 3, wherein the liquid transfer
system is connectable to a vacuum source for selectively creating a
reduced pressure in one of the pair of reaction vessels by means of
at least one gas supply tube.
6. The apparatus according to claim 4, wherein the gas is an inert
gas.
7. The apparatus according to claim 4, wherein the flow of gas is
regulated by at least one valve.
8. The apparatus according to claim 7, further comprising means for
automatically cycling the valve between open and closed
positions.
9. The apparatus according to claim 8, wherein the means for
automatically cycling the valve is a microprocessor.
10. The apparatus according to claim 4, wherein the gas source is
constructed so as to provide the gas flow at a pressure of between
0.1 and 1.0 atmospheres above ambient.
11. The apparatus according to claim 7, comprising means for
automatically controlling the reduction of pressure in the
vessels.
12. The apparatus according to claim 11, wherein the means for
automatically controlling the reduction of pressure is a
microprocessor.
13. The apparatus according to claim 1, further comprising cooling
means positioned between the denaturation vessel and the
polymerization vessel.
14. The apparatus according to claim 13, wherein the cooling means
is a heat exchanger.
15. The apparatus according to claim 1, further comprising means
for adding reagents to at least one of the denaturation vessel and
the polymerization vessel.
16. The apparatus according to claim 1, wherein the means for
adding liquids is a dosing device.
17. The apparatus according to claim 16, wherein the dosing device
is operated by an automatic control device.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an apparatus and a method for
performing automated amplification of nucleic acids under
standardized conditions. The invention also relates to a disposable
part for the apparatus used in the method.
The amplification of nucleic acids is described in U.S. Pat. No.
4,683,194, U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202, and
in patent applications EP 200 362, EP 229 701, EP 237 362 and U.S.
024 604. In amplification, a reaction mixture which contains the
single-stranded target nucleic acid, at least two suitable primers,
four different deoxyribonucleoside triphosphates, and DNA
polymerase in a suitable buffer solution is first incubated at a
suitable temperature in order to polymerize the DNA. Thereafter the
double-stranded DNA formed in the polymerization is denatured by
heating and the reaction mixture is cooled to a temperature at
which the primers are capable of again hybridizing with the target
DNA. When necessary, the temperature of the reaction mixture is
adjusted to a temperature optimal for the action of DNA polymerase,
and DNA polymerase is added. The steps described above are repeated
as many times as is necessary for producing the desired result.
Amplification has in general been performed manually by
transferring the test tubes from one place to another dozens of
times. The method is slow and cumbersome to perform. When
thermolabile polymerase has been used, it has been necessary to
open and close the tubes at intervals. Furthermore, in order to
produce a homogenous reaction fluid, it has been necessary to
centrifuge from the walls of the tubes the reaction fluid condensed
during the cooling step. It has been a further disadvantage that
the reaction conditions vary during repeated steps. This has
constituted a problem, especially when a thermostable enzyme has
been used, since in spite of its thermostability the polymerase is
destroyed if it is kept at a denaturation temperature for too
long.
Patent application EP 236 069 describes an apparatus in which the
amplification of nucleic acids is performed under computer control
by heating and cooling the reaction mixture in the same vessel.
This apparatus has certain disadvantages. It is difficult to heat
and cool a reaction mixture with precision and with sufficient
speed, since in addition to the temperature of the vessel and the
reaction mixture in it, the temperature of the surrounding
apparatus also has to be adjusted. The heat capacity of the
surrounding apparatus is inevitably considerable compared with the
heat capacity of the small-volume (approximately 100 .mu.l)
reaction mixture. For this reason, the reaction mixture is at a
temperature which is disadvantageous for amplification for a
significant portion of each cycle. Problems arise in particular in
the controlling of the denaturation temperature and time. If the
temperature is too low, denaturation will not proceed in the manner
desirable with respect to amplification. On the other hand, if the
polymerase has to be at or near the denaturation temperature for
too long, even thermally stable polymerases will be destroyed.
Thus, in practice, only a thermostable polymerase can be used and
it is active for the duration of only a few reaction cycles.
For the reasons stated above it has not been possible to fully
automate DNA amplification under standardized optimal conditions.
The only automated system to date (described in EP 236,069)
requires the use of a thermostable DNA polymerase and does not
function under optimal conditions.
The object of the present invention is to provide an apparatus and
method eliminating the above-mentioned disadvantages. By using the
apparatus of the present invention it is possible to fully automate
the amplification of nucleic acids in one or several samples
simultaneously under standardized optimal reaction conditions. In
the apparatus according to the invention, the correct denaturation
temperature and time can be adjusted with sufficient precision and
speed so as not to significantly denature thermally stable DNA
polymerase. The method and apparatus according to the invention can
be used for the amplification of nucleic acids regardless of
whether the polymerase used is thermostable or not.
SUMMARY OF THE INVENTION
The invention relates to a method for performing the amplification
of nucleic acids by incubating in a polymerization vessel a
reaction mixture which contains in a suitable buffer solution, one
or several single-stranded target nucleic acids, suitable primers,
deoxyribonucleoside triphosphates and a polymerase. After a
sufficient time for the desired amount of polymerization to occur,
the reaction mixture is transferred into another vessel for the
denaturation of the nucleic acids into single stranded nucleic
acids. After denaturation, the reaction mixture is transferred back
into the original vessel. The amplification process is regulated to
maintain a temperature advantageous for the action of the
polymerization enzyme in the polymerization vessel and a
temperature advantageous for denaturation in the denaturation
vessel. While transferring the reaction mixture from the
denaturation vessel to the polymerization vessel the reaction
mixture may pass through a heat exchanger. In the heat exchanger,
the denatured reaction mixture is preferably cooled to a
temperature at which the primers and the target DNA are capable of
hybridizing.
The invention also relates to an apparatus for use in the method.
The apparatus includes at least one pair of vessels. A vessel pair
is made up of a polymerization vessel and a denaturation vessel.
Each of the vessels is provided with a thermoregulator, for example
a heat block provided with a thermostat, to maintain the
temperature at a level appropriate for polymerization or
denaturation, respectively. Furthermore, the apparatus includes a
liquid transfer system for transferring the reaction liquid from
one vessel to another, which comprises at least one liquid transfer
tube per vessel pair which extends from within the polymerization
vessel to within the denaturation vessel. A preferred liquid
transfer system relies on a pressure differential between the
vessel pair and includes a pair of gas supply tubes.
The apparatus may also be provided with a heat exchanger through
which the liquid transfer tubes may pass, or the liquid transfer
tube may itself serve as a heat exchanger if it is of sufficient
length. Further, the vessel in which the polymerization occurs may
also be equipped with a suitable dosing device for the addition of
polymerase and/or reagents.
The disposable apparatus part of the invention includes, in
packaged combination, a vessel pair and a liquid transfer tube
sized for use in the apparatus of the invention. The vessel pair is
advantageously adapted to receive and form an air tight seal with
the gas tubes supply and dosing tubes of the apparatus, when a
pressure differential liquid transfer system is used.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts an apparatus according to the invention during the
polymerization step of the method of the invention;
FIG. 2 depicts an apparatus according to the invention during the
step of transferring the fluid into the denaturation vessel;
FIG. 3 depicts an apparatus according to the invention during the
step of denaturation of DNA; and
FIG. 4 depicts an apparatus according to the invention during the
transferring of the fluid back into the polymerization vessel.
DESCRIPTION OF THE PREFERRED EMBODIMENT
The method of the invention involves a number of cycles of
polymerization and denaturation in order to produce an amplified
sample from a starting sample containing single stranded nucleic
acids. FIGS. 1 through 4 show one such cycle using an embodiment of
the apparatus of the invention.
As shown in FIGS. 1 through 4, an embodiment of the apparatus of
the invention comprises a polymerization vessel 1 and a
denaturation vessel 2, disposed within thermoregulator means 11 and
12, respectively. Liquid transfer tube 3 extends from the interior
of polymerization vessel 1 to the interior of denaturation vessel 2
and passes through heat exchanger 4. The end of the liquid transfer
tube 3 should in each vessel extend to a point near the bottom of
the vessel so that substantially the entire volume of liquid can be
transferred from one vessel to the next. To optimize liquid
transfer, the vessels will preferably have a substantially conical
base portion extending downward from a substantially cylindrical
top portion as shown in FIGS. 1-4.
Each of the vessels 1 and 2 is also provided with a gas supply tube
5 and 6 through which gases may enter or leave the vessels. The gas
supply tubes 5 and 6 are in turn connected to valves 7 and 8 which
are adapted for connection to a source of gas, preferably an inert
gas such as nitrogen or argon, or vacuum. In the case where a
plurality of vessel pairs are combined in a single apparatus,
manifolds 10 may be interposed between gas supply tubes 5 and 6 and
valves 7 and 8.
Finally, the apparatus shown in FIGS. 1-4 includes a dosing device
9 connected to a dosing tube 9 which is used to provide polymerase
or other reagents to the polymerization vessel 1.
Looking now to the operation of the apparatus in the method of the
invention, FIG. 1 shows the apparatus in a configuration suitable
for the polymerization step. Thus, polymerization vessel 1 contains
a reaction mixture 100 which comprises single stranded target
nucleic acids, suitable primers, polymerase enzyme and nucleoside
triphosphates. The temperature in polymerization vessel 1 is
maintained by thermoregulator means 11 at a level at which
polymerization occurs. During this stage of the method, both valves
7 and 8 are preferably in the deactivated or vent position such
that the interiors of vessels 1 and 2 are at atmospheric
pressure.
After polymerization is substantially complete, the reaction
mixture 100 is transferred from the polymerization vessel 1 to the
denaturation vessel 2 via liquid transfer tube 3. This can be
accomplished by closing valve 7 such that gas flows from tube 14
through valve 7 into polymerization vessel 1 through gas supply
tube 5. Because valve 8 remains in the vent position, reaction
mixture 100 is forced through liquid transfer tube 3 and into the
denaturation vessel 2. Suitable gas pressures are from 0.1 to 1.0
atm above ambient. It will of course be understood that this type
of fluid transfer system requires that the vessels be air tight
when the valves 7 and 8 are in other than the vent position.
After the transfer of reaction mixture is complete, the reaction
mixture 100 is incubated in the denaturation vessel 2 for a period
of time to allow substantially complete denaturation of double
stranded nucleic acids to single stranded nucleic acid. During this
incubation, and indeed throughout the entire process, the
temperature of the denaturation vessel 2 is maintained at a
temperature suitable for denaturation by thermoregulator means 12.
Valves 7 and 8 are again preferably in the vent position to
preclude liquid transfer (FIG. 3).
Finally, after the denaturation step, the reaction mixture 100 is
transferred back to the polymerization vessel 1 via liquid transfer
tube 3 (FIG. 4). Preferably, liquid transfer tube 3 passes through
a heat exchanger 4 which rapidly lowers the temperature to one at
which hybridization of nucleic acids is favored. This allows the
primers and the partially amplified target nucleic acids to be
annealed prior to their return to the polymerization vessel 1 and
is particularly advantageous when the optimum temperature for the
polymerase is not well suited to hybridization. It also prevents
denaturation of polymerase resulting from pouring hot reaction
mixture into the polymerization vessel 1. The transfer of reaction
mixture 100 from denaturation vessel 2 to polymerization vessel 1
is accomplished by closing valve 8 to introduce a flow of gas into
denaturation vessel 2 through gas supply tube 6 while valve 7 is in
the vent position.
The valves which control the flow of gas and thus the transfer of
the reaction mixture between the vessels are preferably
automatically controlled. For example, microprocessors can be used
to control the operation of magnetic three-way valves to make the
amplification process automated, since the steps depicted in FIGS.
1-4 can be repeated several, even tens of times under
microprocessor control. In the preferred embodiment the steps are
repeated a suitable number of times to yield enough amplified DNA
for a specific end use. The amplification therefore occurs
automatically from beginning to end.
The invention is not limited to the embodiment described above and
depicted in FIGS. 1-4 and variations are possible within the scope
of the claimed invention. In the apparatus described above it is
possible to use a vacuum or other means instead of elevated gas
pressure for transfer of the liquids. For example, the reaction
mixture can also be transferred from one vessel to the other by
means of a fluid pump, in which case the gas tubes 5 and 6 are not
necessary. In such a case it is possible to use a pump with a
reversible flow direction, in which case only one liquid transfer
tube 3 is required between the vessels 1 and 2. An amplification
apparatus in which the transfer of liquid is effected by means of
elevated pressure or a vacuum is preferred, however, since it
conveniently enables several target nucleic acid mixtures to be
treated simultaneously in parallel vessel pairs. In such an
apparatus, a single pair of valves can be used for controlling the
transfer of the reaction mixtures in all vessel pairs from one
vessel to the other using manifolds 10 as shown.
It should also be understood that heat exchanger 4 is optional. The
same cooling effect might be achieved by using a longer liquid
transfer tube 3, or may be unnecessary if the polymerization
temperature is suitable for hybridization as well.
The above amplification of nucleic acids can also be performed on
complementary DNA obtained from ribonucleic acid by using a reverse
transcriptase.
The series of amplification reactions is preferably started with
the step according to FIG. 1, i.e. the reagents necessary for the
amplification and a single-stranded target nucleic acid are
incubated at the optimum temperature for the optimum period in
order to perform the polymerase reaction. If the target nucleic
acid is originally double-stranded, it is rendered single-stranded
before the first step. It is, of course, possible to use the
amplification apparatus for the denaturation of the target nucleic
acid, in which case the whole reaction series is started with the
step depicted in FIG. 3. In this case it is in general advantageous
to use a longer denaturation period than during actual
amplification.
The optimum temperatures and times to be used in the reaction
series are determined on the basis of the target nucleic acid, as
well as the primer and polymerase used. A person skilled in the art
is able to adjust the apparatus and to select the suitable
conditions for the amplification reactions to be performed at a
given time.
A number of different procedures are possible with respect to the
addition of the polymerase. The enzyme may be introduced either
continuously or intermittently by automatic means into the
polymerase reaction vessel. If the enzyme is thermostable, it is
incorporated into the reaction mixture at the beginning of the
reaction series and added thereafter only when needed. No enzyme
needs to be added if it has been introduced into the polymerase
reaction vessel either in an immobilized form or in a suitable
slowly releasing dosage form. If the enzyme is immobilized, it of
course does not pass from the reaction vessel. When a slowly
releasing dosage form is used, it is important that the release of
the polymerase is regulated so that its concentration remains
suitable for the duration of as many reaction cycles as is
necessary.
The temperature of the heat exchanger is determined by the
hybridization temperature of the primer to the single-stranded DNA.
When the hybridization temperature of the primer differs
considerably from the optimum temperature of the polymerization
enzyme, the temperature profile of the reaction mixture is
preferably regulated so that the primer and single-stranded DNA
have time to hybridize in the heat exchanger. The thermolability of
the polymerization enzyme can also be taken into consideration in
the regulation of the heat exchanger by making sure that the
reaction mixture is cooled sufficiently before it comes into
contact with the enzyme. The retention time in the heat exchanger
of the solution which contains the denatured nucleic acid can be
regulated by means of the tube length and the pressure of the gas
introduced into the denaturation vessel, or respectively by
adjusting the vacuum or the efficiency of the fluid pump.
* * * * *